The present invention relates to sample introduction and management, in particular in a high performance liquid chromatography application such as one-dimensional or two-dimensional HPLC.
In high performance liquid chromatography (HPLC), a liquid has to be provided usually at a very controlled flow rate (e. g. in the range of microliters to milliliters per minute) and at high pressure (typically 20-100 MPa, 200-1000 bar, and beyond up to currently 200 MPa, 2000 bar) at which compressibility of the liquid becomes noticeable. For liquid separation in an HPLC system, a mobile phase comprising a sample fluid (e.g. a chemical or biological mixture) with compounds to be separated is driven through a stationary phase (such as a chromatographic column packing), thus separating different compounds of the sample fluid which may then be identified. The term compound, as used herein, shall cover compounds which might comprise one or more different components.
The mobile phase, for example a solvent, is pumped under high pressure typically through a chromatographic column containing packing medium (also referred to as packing material or stationary phase). As the sample is carried through the column by the liquid flow, the different compounds, each one having a different affinity to the packing medium, move through the column at different speeds. Those compounds having greater affinity for the stationary phase move more slowly through the column than those having less affinity, and this speed differential results in the compounds being separated from one another as they pass through the column. The stationary phase is subject to a mechanical force generated in particular by a hydraulic pump that pumps the mobile phase usually from an upstream connection of the column to a downstream connection of the column. As a result of flow, depending on the physical properties of the stationary phase and the mobile phase, a relatively high pressure drop is generated across the column.
The mobile phase with the separated compounds exits the column and passes through a detector, which registers and/or identifies the molecules, for example by spectrophotometric absorbance measurements. A two-dimensional plot of the detector measurements against elution time or volume, known as a chromatogram, may be made, and from the chromatogram the compounds may be identified. For each compound, the chromatogram displays a separate curve feature also designated as a “peak”. Efficient separation of the compounds by the column is advantageous because it provides for measurements yielding well defined peaks having sharp maxima inflection points and narrow base widths, allowing excellent resolution and reliable identification and quantitation of the mixture constituents. Broad peaks, caused by poor column performance, so called “Internal Band Broadening” or poor system performance, so called “External Band Broadening” are undesirable as they may allow minor components of the mixture to be masked by major components and go unidentified.
Two-dimensional separation of a fluidic sample denotes a separation technique in which a first separation procedure in a first separation unit is performed to separate a fluidic sample into a plurality of fractions, and in which a subsequent second separation procedure in a second separation unit is performed to further separate the plurality of fractions into sub-fractions. Two-dimensional liquid chromatography (2D LC) may combine two liquid chromatography separation techniques. When performing a 2D LC measurement, operation of two pumps needs to be coordinated in itself and with the action of further system components managing the sample and fraction transport, for instance with correspondingly switching fluidic valves. The sample and fraction pathway switching may result in pressure ripples or dips acting on separation units and other components of the fluid separation system, thereby deteriorating the chromatographic performance, reliability of the system and longevity of its components.
In so-called Comprehensive 2D LC, all eluate coming from the first dimension (e.g. the entire solvent flow containing the sample components past separation in the first chromatographic column) is coupled into the second dimension and further separated there. This significantly increases the requirements for processing speed in the second dimension. Typically, the solvent flow (containing the sample components) is fed into the second dimension in portions (also referred to as “sniplets”). Cycle times for processing of a single sniplet can be as fast as 15 seconds or lower. In such case with 4 cycles per minute, 24 hours of continued work means 5760 modulations or sample injections, which may come close to the lifetime of a column under usual operation conditions. In a number of arrangements the first dimension, or generally a sniplet source, provides sample contained in a solvent which may be too strong for the downstream dimension (e.g. for the second dimension). This can occur, e.g., when using HILIC in first dimension and RP (reversed phase) chromatography in second dimension, so it may be of advantage to dilute the sample plug (sniplet) coming from the collection loop before it hits the column.
Columns can be sensitive to flow disruptions and e.g. to reconnections with sample loops which hold lower pressure than the column itself (flow reversal due to backwards de-compression of the column content). Also the column may be sensitive to abrupt (re)connection to high pressure sources, resulting in pressure shocks on the column and packing material deterioration. These can be substantial factors of column aging, wear and deterioration.
Sample introduction in HPLC systems is disclosed e.g. in U.S. Pat. No. 3,916,692A, WO2006083776A2, U.S. Pat. No. 8,047,060B2.
WO2012175111A1, by the same applicant, discloses a two-dimensional HPLC system.